Concentrating a target molecule for sensing by a nanopore

ABSTRACT

Methods and related products are disclosed that improve the probability of interaction between a target molecule and a nanopore by capturing the target molecule on a surface comprising the nanopore. The captured target molecule, the nanopore, or both, are able to move relative to each other along the surface. When the leader of the target molecule is in proximity with the nanopore, interaction of the target portion of the target molecule with the nanopore occurs, thereby permitting sensing of the target portion. Confining the target molecule and nanopore in this manner leads to significantly enhanced interaction with the nanopore.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 870225_408_SEQUENCE_LISTING.txt. The text fileis 14.2 KB, was created on Sep. 1, 2015, and is being submittedelectronically via EFS-Web.

BACKGROUND

Technical Field

This invention is generally directed to concentrating a target moleculefor sensing by a nanopore, as well as methods and products relating tothe same.

Description of the Related Art

Measurement of biomolecules is a foundation of modern medicine and isbroadly used in medical research, and more specifically in diagnosticsand therapy, as well in drug development. Nucleic acids encode thenecessary information for living things to function and reproduce, andare essentially a blueprint for life. Determining such blueprints isuseful in pure research as well as in applied sciences. In medicine,sequencing can be used for diagnosis and to develop treatments for avariety of pathologies, including cancer, heart disease, autoimmunedisorders, multiple sclerosis, and obesity. In industry, sequencing canbe used to design improved enzymatic processes or synthetic organisms.In biology, this tool can be used to study the health of ecosystems, forexample, and thus have a broad range of utility. Similarly, measurementof proteins and other biomolecules has provided markers andunderstanding of disease and pathogenic propagation.

An individual's unique DNA sequence provides valuable informationconcerning their susceptibility to certain diseases. It also providespatients with the opportunity to screen for early detection and/or toreceive preventative treatment. Furthermore, given a patient'sindividual blueprint, clinicians will be able to administer personalizedtherapy to maximize drug efficacy and/or to minimize the risk of anadverse drug response. Similarly, determining the blueprint ofpathogenic organisms can lead to new treatments for infectious diseasesand more robust pathogen surveillance. Low cost, whole genome DNAsequencing will provide the foundation for modern medicine. To achievethis goal, sequencing technologies must continue to advance with respectto throughput, accuracy, and read length.

Over the last decade, a multitude of next generation DNA sequencingtechnologies have become commercially available and have dramaticallyreduced the cost of sequencing whole genomes. These include sequencingby synthesis (“SBS”) platforms (Illumina, Inc., 454 Life Sciences, IonTorrent, Pacific Biosciences) and analogous ligation based platforms(Complete Genomics, Life Technologies Corporation). A number of othertechnologies are being developed that utilize a wide variety of sampleprocessing and detection methods. For example, GnuBio, Inc. (Cambridge,Mass.) uses picoliter reaction vessels to control millions of discreetprobe sequencing reactions, whereas Halcyon Molecular (Redwood City,Calif.) was attempting to develop technology for direct DNA measurementusing a transmission electron microscope.

Nanopore based nucleic acid sequencing is a compelling approach that hasbeen widely studied. Kasianowicz et al. (Proc. Natl. Acad. Sci. USA 93:13770-13773, 1996) characterized single-stranded polynucleotides as theywere electrically translocated through an alpha hemolysin nanoporeembedded in a lipid bilayer. It was demonstrated that duringpolynucleotide translocation partial blockage of the nanopore aperturecould be measured as a decrease in ionic current. Polynucleotidesequencing in nanopores, however, is burdened by having to resolvetightly spaced bases (0.34 nm) with small signal differences immersed insignificant background noise. The measurement challenge of single baseresolution in a nanopore is made more demanding due to the rapidtranslocation rates observed for polynucleotides, which are typically onthe order of 1 base per microsecond. Translocation speed can be reducedby adjusting run parameters such as voltage, salt composition, pH,temperature, and viscosity, to name a few. However, such adjustmentshave been unable to reduce translocation speed to a level that allowsfor single base resolution.

Stratos Genomics has developed a method called Sequencing by Expansion(“SBX”) that uses a biochemical process to transcribe the sequence ofDNA onto a measurable polymer called an “Xpandomer” (Kokoris et al.,U.S. Pat. No. 7,939,259, “High Throughput Nucleic Acid Sequencing byExpansion”). The transcribed sequence is encoded along the Xpandomerbackbone in high signal-to-noise reporters that are separated by ˜10 nmand are designed for high-signal-to-noise, well-differentiatedresponses. These differences provide significant performanceenhancements in sequence read efficiency and accuracy of Xpandomersrelative to native DNA. Xpandomers can enable several next generationDNA sequencing detection technologies and are well suited to nanoporesequencing.

Gundlach et al. (Proc. Natl. Acad. Sci. 107(37): 16060-16065, 2010) havedemonstrated a method of sequencing DNA that uses a low noise nanoporederived from Mycobacterium smegmatis (“MspA”) in conjunction with aprocess called duplex interrupted sequencing. In short, a double strandduplex is used to temporarily hold the single stranded portion in theMspA constriction. This process enables better statistical sampling ofthe bases held in the limiting aperture. Under such conditions singlebase identification was demonstrated; however, this approach requiresDNA conversion methods such as those disclosed by Kokoris et al.(supra).

Akeson et al. (WO2006/028508) disclosed methods for characterizingpolynucleotides in a nanopore that utilize an adjacently positionedmolecular motor to control the translocation rate of the polynucleotidethrough or adjacent to the nanopore aperture. At this controlledtranslocation rate (350-2000 Hz (implied measurement rate)), the signalcorresponding to the movement of the target polynucleotide with respectto the nanopore aperture can be more closely correlated to the identityof the bases within and proximal to the aperture constriction. Even withmolecular motor control of polynucleotide translocation rate through ananopore, single base measurement resolution is still limited to thedimension and composition of the aperture constriction. As such, inseparate work, Bayley et al. (alpha hemolysin: Chemistry & Biology9(7):829-838, 2002) and Gundlach et al. (MspA: Proceedings of theNational Academy of Sciences 105(52):20647-20652, 2008) have disclosedmethods for engineering nanopores with enhanced noise and baseresolution characteristics. However, a demonstration of processiveindividual nucleotide sequencing has yet to be published that useseither (or both) a molecular motor for translocation control and anengineered nanopore. Current state of the art suggests that signaldeconvolution of at least triplet base sets would be required in orderto assign single base identity.

Nanopores have proven to be powerful amplifiers, much like their highlyexploited predecessors, Coulter Counters. However, a limitation of thesedevices is their limit of detection. High concentrations of samplematerials are required for rapid detection because the ends of longnucleic acid molecules are statistically challenged to find the nanoporeentry. Branton et al. (Nat Biotech 26(10):1146-1153, 2008) calculatedthat 10⁸ full genomes would be required to adequately sequence a genomebased upon extrapolated throughput. Indeed, improving the limit ofdetection for many biomolecular measurements is highly desirable forimproving sensitivity and extending the range of applications.

While significant advances have been made in this field, there remains aneed in the art for new and improved methods and materials for enhancingbiomolecular interactions and/or measurements. The present inventionfulfills these needs and provides further related advantages.

BRIEF SUMMARY

In brief, a method is disclosed for concentrating a target molecule fornanopore sensing, comprising capturing the target molecule on a surfacecomprising a nanopore and a hydrophobic domain. The target moleculecomprises a target portion, a hydrophobic capture element and a leaderfor interaction with the nanopore. The hydrophobic capture element ofthe target molecule is associated with, and capable of movement along,the hydrophobic domain of the surface to bring the leader of the targetmolecule in proximity with the nanopore. At least the target portion ofthe target molecule is sensed by the nanopore upon interaction with thenanopore.

In one embodiment, the step of capturing the target molecule on thesurface comprises contacting the surface with the target molecule,wherein the target molecule comprises, prior to the contacting step, thetarget portion, the hydrophobic capture element and the leader.

In another embodiment, the step of capturing the target molecule on thesurface comprises linking the hydrophobic capture element associatedwith the surface to the target portion and leader, thereby capturing thetarget molecule on the surface.

In a more specific embodiment, the nanopore is a biological nanopore.

In a more specific embodiment, the surface is a lipid bilayer, asolid-state and/or synthetic membrane.

In a more specific embodiment, the target portion comprises nucleicacids, a linear polymer, a molecular bar code and/or an Xpandomer.

In a more specific embodiment, the leader is a hydrophilic polymer.

In a more specific embodiment, the hydrophobic capture element is analiphatic hydrocarbon.

In a more specific embodiment, the target molecule comprises two or morehydrophobic capture elements.

These and other aspects of the invention will be evident upon referencesto the attached drawings and following detailed description.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 illustrates capture of several target molecules on a surfacecomprising a nanopore, as well as translocation of a target moleculethrough a nanopore.

FIG. 2 illustrates translocation of a target molecule through ananopore, wherein the target molecule has multiple hydrophobic captureelements.

FIG. 3 illustrates linking the leader and target portion of the targetmolecule to the hydrophobic capture element on a surface, as well astranslocation of the leader and target portion of the target molecule(but not the hydrophobic capture element) through a nanopore.

FIG. 4A illustrates relative event capture in a nanopore due to endmodifications of the targeted molecule. FIG. 4B shows a target molecule(SEQ ID NO: 5) that has 4 duplexed regions (SEQ ID NOS: 1-4,respectively) used to pause and measure the molecule in a nanopore. Theend-modifications (SEQ ID NOS: 25-27, respectively) (^(3′)×) are shownbelow the target molecule in FIG. 4B.

FIG. 5 illustrates the structure of a control (SEQ ID NO: 6) and atarget molecule (SEQ ID NO: 7) used to assess the concentrationenhancement caused by different end modifications. Structures of fivedifferent end modifications (Y^(3′)) (SEQ ID NOS: 8-12, respectively)are shown below the target molecule structure, which have hydrophobicgroups of different sizes and a fixed leader length.

FIG. 6 illustrates the structures of four additional end modifications(SEQ ID NOS: 13-16, respectively) that have a fixed hydrophobic groupsize and leaders of different sizes.

FIG. 7 illustrates the structures of four hydrophobic capture elements(SEQ ID NOS: 17-20, respectively) which are designed to hybridize to thetarget molecule (rather than part of the covalent structure).

FIG. 8 illustrates structures of three end-adapted ds-DNA targets (SEQID NOS: 21-23, respectively) used to compare different leaders (SEQ IDNOS: 24).

FIG. 9 illustrates concentrating a target molecule utilizing asolid-state nanopore with supported lipid bilayers.

FIG. 10A illustrates the distribution of adapted molecules that areassociated with the lipid bilayer and can freely diffuse along the planeof the bilayer. FIG. 10B shows how a shear force, such as flow, candrive such adapted targets to concentrate and localize at a nanoporenear the edge of the bilayer plane.

DETAILED DESCRIPTION

In brief, the invention improves the probability of interaction betweena target molecule and a nanopore by capturing the target molecule on asurface comprising the nanopore. The captured target molecule, thenanopore, or both, are able to move relative to each other along thesurface. In this way, the volume occupied by the target molecule and thenanopore is dramatically reduced compared, for example, to a targetmolecule in a volume of solution that is in contact with the surface. Byconfining the target molecule and nanopore in this manner—also referredto herein as “concentrating” the target molecule—the probability ofinteraction between the target molecule and the nanopore issignificantly increased. Such increased concentration leads tosignificantly enhanced translocation of the target molecule, or targetportion thereof, through the nanopore.

Nanopores may be broadly classified into two types, biological andsynthetic, and both types are intended to be within the scope of thisinvention. While alpha hemolysin (αHL) is perhaps the most studiedbiological nanopore to date, this and other over biological nanoporesmay be utilized in the context of this invention, such as mycobacteriumsmegmatis porin A (MspA). More recently, synthetic nanopores have beenintroduced using polymers, glass and thin solid-state membranes. Again,all such design options are within the scope of this invention.

Nanopores are, in effect, small holes through a surface. In the case ofbiological nanopores, the surface is typically a membrane such as alipid bilayer. However, other surfaces may also be employed, includinglipid monolayers or oil/water interfaces, as well as synthetic and/orinorganic membranes. In the practice of this invention, the surfacecomprises the nanopore, and also comprises a hydrophobic domain. In thecase of a lipid bilayer in aqueous media, for example, the hydrophobicdomain is located in the interior portion (i.e., where the hydrophobictails of the phospholipids lie). In addition to lipid bilayers, otherhydrophobic/hydrophilic interfaces can be used for the surface,including (for example) an oil/water interface, a tethered lipid/waterinterface, an air/water interface, or a lipid-hydrophobicsubstrate/water interface. In general, these surfaces exhibitdifferential hydrophobicity and enable capture of the hydrophobiccapture element of the target molecule. In addition, such surfaces donot spatially fix the captured target molecule at a given location onthe surface, but instead allow the target molecule to diffuse along thesurface.

As mentioned above, the target portion may comprise, for example,nucleic acids or a linear polymer. In another embodiment, the targetportion may comprise a molecular bar code such as taught in Akeson etal. (U.S. Pat. No. 6,465,193), and/or an Xpandomer such as taught inKokoris et al. (supra).

The hydrophobic capture element of the target molecule is associatedwith the hydrophobic domain of the surface. As used herein, associatedmeans that the hydrophobic capture element of the target molecule andthe hydrophobic domain of the surface cause the target molecule toremain joined to the surface, while also permitting the captured targetmolecule to move along the hydrophobic domain of the surface to bringthe target molecule in proximity with the nanopore. Suchhydrophobic-hydrophobic interaction is mostly an entropic effectassociated with disruption of highly dynamic hydrogen bonds betweenwater molecules and nonpolar substances. The strength of hydrophobicinteractions depends on temperature, as well as the shape and number ofcarbon atoms on the hydrophobic compound.

As mentioned above, the target molecule comprises a target portion, ahydrophobic capture element, and a leader. In one embodiment, thesurface is contacted with the target molecule such that the captureelement of the target molecule is associated with the hydrophobic domainof the surface, thereby capturing the target molecule. In an alternativeembodiment, the surface having the hydrophobic capture elementassociated therewith is contacted with the target portion and leader,thereby capturing the target molecule on the surface.

Once captured by the surface, the leader portion of the target moleculeis capable of interacting with the nanopore in a manner that promotesinteraction of the target molecule (or target portion thereof) with thenanopore. Such interaction includes, for example, complete or partialtranslocation through the nanopore. Other interactions may involvepositioning a target protein at the nanopore for measurement, or toposition a functional protein, such as an enzyme, proximal to thenanopore. Typically, the leader is not hydrophobic, and in oneembodiment is a hydrophilic (charged) polymer of low mass to allowinteraction with the nanopore when the nanopore and the leader of thetarget molecule are in close proximity. As mentioned above, the capturedtarget molecule, the nanopore, or both, are capable of movement relativeto each other along the surface.

Concentrating the target molecule in this manner increases the number ofinteractions of the target molecule (or target portion thereof) with thenanopore. As an illustrative example, one application of this inventionrelates to increasing the number of complete or partial translocationsof the target portion, such as DNA/RNA, through a nanopore, wherein theDNA/RNA target portion is combined with a hydrophobic capture elementand an oligomer leader. In this representative example, the hydrophobiccapture element is captured in the hydrophobic domain of the lipidbilayer that supports the nanopore. However, the target molecule stillmaintains lateral mobility across the lipid bilayer surface. Thisincreases the probability that the oligomer leader will be drawn intothe nanopore and increases the frequency of DNA/RNA translocationthrough the nanopore.

While nanopores have traditionally been developed for nucleic acidanalysis, the target portion of the target molecule may be any of avariety of polymeric materials suitable to measurement and/or detectionby the nanopore. In one example, the target portion is an Xpandomer asdisclosed in WO2008/157696 (U.S. Pat. No. 7,939,259), as well as relatedembodiments as disclosed in WO2009/055617, WO2010/088557 andWO2012/003330 (each of which are hereby incorporated by reference intheir entirety). For example, Xpandomers synthesized from ligation-basedextension of hexamer Xprobes have been end-adapted with C-48-polyA₂₅leaders and have demonstrated translocation rates of 3 events per minutewith addition of 10 fmol of material. In this embodiment, the C-48portion is a concatenate of 4 dodecyl phosphodiester monomers and actsas the hydrophobic capture element, while the polyA₂₅ portion is a 25base deoxyadenosine homopolymer that functions as the leader element.Under identical conditions, the same Xpandomers adapted to polyA₂₅leaders required additions of 1 pmol for the same event rate. In bothcases the nanopore was wild-type alpha-hemolysin embedded in a 13 microndiameter lipid bilayer.

In one embodiment, as illustrated in FIG. 1, target molecule 120comprises target portion 150, hydrophobic capture element 160 and leader170 which, in this figure, is shown having substantially translocatedthrough nanopore 110 in surface 130. The direction of translocationthrough the nanopore is shown by arrow 115. In addition to targetmolecule 120, FIG. 1 also depicts target molecules 121, 122 and 123having hydrophobic capture elements 161, 162, 163, respectively,captured by the hydrophobic domain of surface 130, which in this figureis depicted as the interior (hydrophobic) portion of a lipid bilayer.Captured target molecules 121, 122 and 123 further comprise targetportions 151, 152 and 153 and leaders 171, 172 and 173, respectively.The dots (“• • •”) shown at the ends of target portions 151, 152 and 153represent additional length of the target portion. Captured targetmolecules 121, 122 and 123 are capable of movement along surface 130 (asdepicted by arrow 116), and such movement brings the leader of acaptured target molecule in proximity with the nanopore, as depicted byleader 171 of target molecule 121 being near nanopore 110. Suchproximity allows the leader to interact with the nanopore, thus drawingthe target molecule into the nanopore for translocation as depicted bytarget molecule 120.

In a more specific embodiment of FIG. 1, the hydrophobic capture elementis a C48 aliphatic hydrophobic group and the leader is polyA₂₄ oligomerthat acts as a hydrophilic polyanionic leader. The sample reservoir has1 M potassium chloride in an aqueous 10 mM HEPES pH 7.4 buffer. As thetarget molecule diffuses through the reservoir, it eventually interactswith the lipid bilayer and the hydrophobic capture element embeds intothe hydrophobic portion of the lipid bilayer core. The target moleculeis now captured by the surface and the hydrophilic leader is localizedin the reservoir close to the surface of the lipid bilayer. Multipletarget molecules concentrate on the lipid bilayer in this manner anddiffuse along the surface until the leader of the target molecule isproximal to the nanopore. An electric field acting across the poreapplies a force on the negatively charged leader, drawing it through thepore and pulling the hydrophobic capture element free of the lipid sotranslocation of the remainder of the target molecule can proceed. Inthis manner, the rate of capture and translocation is increased byorders of magnitude relative to the corresponding target portion insolution interacting with the nanopore.

In another embodiment, as illustrated in FIG. 2, the target moleculecomprises more than one hydrophobic capture element. In particular, FIG.2 illustrates a target molecule having four hydrophobic capture elements261, 262, 263 and 264. Hydrophobic capture elements 261, 262 and 263 areshown as captured by the hydrophobic domain of surface 230, which inFIG. 2 is depicted as a lipid bilayer. Target molecule 220 is shown inFIG. 2 as having partially translocated through nanopore 210 in thedirection of arrow 215. Hydrophobic capture element 264 is depicted ashaving already been dislodged from the hydrophobic domain of surface230, and is in the process of translocating through the nanopore.

In a more specific embodiment of FIG. 2, a control molecule was preparedhaving six ligated heterogeneous polymer units, with each polymer unithaving four PEG-6 (hexaethyleneglycol phosphodiester) with anamino-modified base. One end of the polymer was adapted with a poly-A₅₀oligomer (forming the leader) (270). The structure on the other end ofthe polymer is a hairpin loop (290) that is used to prevent backwardentry into the pore. The hairpin loop is too large to enter the porefirst, but when the hairpin loop is pulled through at the end, theduplex portion will open and unfold the loop, allowing it totranslocate. This control molecule was compared to a target moleculewhich is identical except that each pendant amino group (of theamino-modified base) was conjugated with a DiBenzoCycloOctyl (DBCO)hydrophobic moiety (forming the hydrophobic capture element). For theresulting target molecule, the DBCO moieties interact with thehydrophobic interior of the lipid bilayer, thus capturing the targetmolecule on the surface, and thereby increasing the concentration of theleader (the poly-A₅₀ segment) near the nanopore. Having the leader inclose proximity with the nanopore increases the probability that thetarget molecule will be translocated through the nanopore.

Translocation frequency through the nanopore (alpha hemolysin) of thetarget molecule compared to the control molecule showed increases of 30,15, 9, 10 and 8 times for applied potentials of 100, 110, 120, 130 and140 mV, respectively. For these measurements, the cis and transreservoirs had 2.0 M LiCl, 10 mM HEPES, pH of 7.4 at a temperature of10° C. and 15 pmol of control or target molecule was added to the 100 μlcis reservoir. The nanopore was a wild-type α-hemolysin (Sigma Aldrich)and the lipid bilayer was formed on a 13 micron diameter teflon aperturewith 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (Avanti PolarLipids) lipid bilayer. (Such methods follow those described by Jetha etal., Chapter 9. Micro and Nano Technologies in Bioanalysis, Humana Press2009, which is incorporated herein by reference.)

In FIGS. 1 and 2 discussed above, capturing the target molecule on thesurface comprises contacting the surface with the target molecule,wherein the target molecule comprises the target portion, thehydrophobic capture element and the leader prior to the capturing step.In another embodiment, capturing the target molecule on the surfacecomprises contacting the surface with the hydrophobic capture elementand linking the hydrophobic capture element to the target portion andleader to yield the captured target molecule on the surface.

Accordingly, and in another embodiment as illustrated in FIG. 3,hydrophobic capture element 361 is captured by the hydrophobic domain ofsurface 330, which in this figure is depicted as a lipid bilayer. Thehydrophobic element 361 may be captured during the formation of surface330 or may be captured after surface 330 is formed. Hydrophobic captureelement 361 further comprises linking element 381 which permits linkageof hydrophobic capture element 361 to leader 371 and target portion 351by attachment to corresponding linking element 382. Upon linkage of thecapture element to the leader and target portion, target molecule 328 isboth formed and captured on the surface. Once captured, the targetmolecule is capable of diffusing along the surface until the leader ofthe target molecule is proximal to nanopore 310, as depicted by targetmolecule 328 in FIG. 3. Such proximity allows the leader to interactwith the nanopore, thus drawing the leader and target portion of thetarget molecule (shown as leader/target portion 325) into the nanoporefor translocation there through.

In a more specific embodiment of FIG. 3, capture element 361 comprisesan aliphatic group and an oligodeoxynucleotide (ODN) linker. Thealiphatic group (the hydrophobic capture element) remains embeddedwithin the hydrophobic domain of surface 330, which is shown as a lipidbilayer, and the ODN linker extends out of the lipid bilayer and intothe aqueous. In this embodiment the leader and target portion areadapted with a nucleic acid segment 382 complementary to ODN linker 381.This linker pair is used to join (by hybridization) the leader andtarget portion to the hydrophobic capture element, thereby forming thetarget molecule at the surface, as depicted by target molecule 328. Thealiphatic group (hydrophobic capture element) diffuses freely throughoutthe plane of the lipid bilayer. This localization to the plane of thelipid bilayer increases the probability of interaction between thecaptured target molecule and the nanopore, resulting in the leader beingelectrophoretically drawn into the nanopore. During translocation,either the linker releases (e.g., the hybridized linkage unzips), or thelinker remains attached and the hydrophobic capture element is pulledfree of the lipid bilayer and is stripped off at the nanopore.

A representative example of such a hydrophobic capture element isdisclosed by Chan et al. (Proceedings of the National Academy ofSciences 106(4): 979-984, 2009), which discloses the synthesis of ahydrophobic capture element inserted into a lipid bilayer and linked toa vesicle. In this case, the hydrophobic portion of the capture elementwas one of the lipid molecules that forms the lipid bilayer, and thislipid molecule was conjugated to an ODN linker. The ODN linker, in turn,was used to hybridize to a complement ODN that was conjugated to avesicle, demonstrating capture of the vesicle. In another example,Grenali et al. (Langmuir 22(1):292-299, 2006) showed that bilayers where0.5% of the lipids were head-adapted with biotin followed by neutravidinwould capture biotinylated oligonucleotides. These capturedoligonucleotides would freely diffuse along the bilayer surface with adiffusion constant 26% of that for the lipids themselves.

The hydrophobic capture element may be controlled in size to facilitatediffusive capture of the target molecule with limited diffusive releasefrom the surface, such as a lipid bilayer. However, it should alsorelease with sufficient ease and be sized such that translocation is notinterrupted. In one embodiment, a single length of an aliphatic elementthat is in-line with the backbone of the target molecule may beutilized. If the length of the aliphatic element is too short, thehydrophilic portions of the target molecule (such as the leader) willlimit its interaction with the lipid bilayer's hydrophobic core. Thus,the hydrophobic capture element should be large enough to resist theentropic force that the target molecule will exert. However, if thehydrophobic capture element is too long, translocation may be limiteddue to reduced target molecule mobility in the lipid bilayer; namely,the electrophoretic force required to promote translocation could exceedoptimum run conditions and reduce measurement quality. In addition,excessively long hydrophobic segments may cause target handling issues(particularly in an aqueous environment) and have a disruptive effect onlipid bilayer stability. To increase the capture strength of thehydrophobic capture element while maintaining shorter lengths, thetarget molecule may contain additional (i.e., more than one) hydrophobiccapture elements. Also, embodiments other than linear in-line geometriesmay be utilized, such as hydrophobic capture elements pendent orbranched off the target molecule backbone.

In a further embodiment, the hydrophobic capture element may be modifiedin order to selectively pause translocation through the nanopore, asillustrated by the data presented in the bar graph of FIG. 4A. In thisexperiment, translocation frequencies were measured for the linearpolymer of FIG. 4B with 4 nucleic acid duplexes having total contourlength of ˜45 nm (i.e., the target portion). Using the translocationcontrol method described by Akeson et al. (supra) and Gundlach et al.(supra), the duplexes are used to pause the polymer translocation for aperiod of time sufficient to measure a distinct current blockage level.The blockage level is determined principally by the duplex at thenanopore entrance and the portion of polymer that threads the nanoporebarrel. After a stochastic pause, the duplexes are stripped off thepolymer backbone and the polymer translocation proceeds until it ispaused by the next duplex portion. This polymer uses the same 14base-pair duplex but alternates with threaded portions DDDDAAA or DDDDD,where “D” represents a hexaethyleneglycol phosphodiester linked monomerand “A” is an adenosine deoxynucleotide. Translocation of the moleculecan be determined from a characteristic signature of 4 levelsalternating between current blockage of 0.31 and 0.18 (relative to openpore current). Measurement was made at 20° C., 120 mV, and 1M KCl/10 mMHEPES/pH7.4 buffer. The nanopore was a wild-type α-hemolysin (SigmaAldrich) and the lipid bilayer was formed on a 13 micron diameter teflonaperture with 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (AvantiPolar Lipids) lipid bilayer. The methods follow those described by Jethaet al. (Chapter 9. Micro and Nano Technologies in Bioanalysis, HumanaPress 2009).

The 3′ end of the target portion was linked to one of three groups: (1)polyA₅₀ (SEQ ID NO: 25); (2) C48-polyA₂₅ (SEQ ID NO: 6) or (3)C60-polyA₂₅. (SEQ ID NO: 27). C48 and C60 are carbon chains of 48 and 60carbons, respectively, synthesized from dodecyl phosphodiester linkedmonomers. For example, 5 of the 12-carbon monomers may be linked to forma C60 (the phosphate linkage between such C12 monomers is anionic andwill moderate the hydrophobicity of the C12 concatenate to some degree).For polymer (1), polyA₅₀ (SEQ ID NO: 25) served as the leader to thetarget portion (without hydrophobic capture element). For polymers (2)and (3), the C48 and C60 segments, respectively, served as thehydrophobic capture elements, while polyA₂₅ served as the leader.

Control polymer (1) (i.e., target portion joined to leader withouthydrophobic capture element) and target molecules (2) and (3) weremeasured for translocation frequency through a nanopore. As shown in thebar chart of FIG. 4A, target molecule (2) (C48-polyA₂₅) (SEQ ID NO: 26)and (3) (C60-polyA₂₅) (SEQ ID NO:27) had significantly enhancedfrequency of translocation events compared to comparative polymer (1)(polyA₅₀)(SEQ ID NO: 25) In particular, in relation to the comparativepolymer (1), target polymer (2) increased the number of translocationevents/min/pmol by 920 times under the same experimental conditions.

It should be noted that the data presented in FIG. 4A were captured onindependent runs and the effective measurement time (due to nanoporeblockages) varied between runs. Samples were introduced to the 100microliter Cis reservoir of the nanopore in a 2 microliter aliquotloaded with 15 femtomoles of sample. To maximize translocation ratesfrom the small sample size, the sample was injected directly adjacent tothe nanopore, maximizing the sample interaction with the nanopore, butresults often varied by factors of 5 or more. Despite these variations,the concentrator method consistently gave higher translocation rateswhen compared to non-hydrophobic capture sample

To reduce sample injection variations, a control molecule was mixed witheach target molecule tested. Nanopore translocations of the target andcontrol could be distinguished by their unique sequence of currentblockage signals using the duplex translocation control method describedabove. The results that follow utilize this approach and were derivedfrom measurements made at 20° C. and 130 mV. The Trans well solutionused for these measurements was 2M NH4Cl buffered with 10 mMHEPES/pH7.4; the Cis well solution was 0.4M NH4Cl/0.6M Guanidine HClbuffered with 10 mM HEPES/pH7.4. The nanopore was a wild-typeα-hemolysin (Sigma Aldrich) and the lipid bilayer was formed on a 13micron diameter teflon aperture generally using1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (Avanti Polar Lipids)lipid bilayer. In all cases duplexes are added to the target or controlin excess of the number of binding sites by a factor of 100× and arethermally cycled.

Test molecules were synthesized on a Mermaide 12 oligonucleotidesynthesizer (BioAutomation, Texas) using a variety of phosphoamiditeslisted at the bottom of FIG. 5. In some cases, longer molecules wereformed from two parts that are enzymatically ligated to make the fullconstruct. FIG. 5 shows the composition of the target and controlmolecules. Each had six duplexed regions that provided a differentblockage level sequence when measured in the nanopore.

Referring to FIG. 5, two types of 6-base duplexes are shown adjacent totheir complementary sites along the target and control molecules(^(3′)AGKCKG^(5′) and ^(3′)ATKGKT^(5′)); each use a modified base-typecalled a G-Clamp (Glen Research, Sterling, Va., represented as “K”) toprovide stronger duplexing. This experiment compared translocation ratesof the target molecule with five different end-adaptations; namely,dA₆C₃₆dA₂₄ (SEQ ID NO: 8), dA₅C₄₈dA₂₄ (SEQ ID NO: 9), dA₄C₆₀dA₂₄ (SEQ IDNO: 10), C₁₀₈dA₂₄ (SEQ ID NO: 11) and dA₉dA₂₄ (SEQ ID NO: 12). The firstfour targets had aliphatic segments of different lengths (hydrophobiccapture elements) and the latter end-adaptation had no aliphaticsegment. In each case the distal dA₂₄ was the leader. The controlmolecule was end-adapted with dA₅C₄₈dA₂₄ (SEQ ID NO: 9), and was mixedat equal concentration with one of the target species. For eachmeasurement, a 2 microliter aliquot containing 150 femtomoles of eachwas injected adjacent to the nanopore and measured. Translocation eventswere captured and discriminated to calculate translocation rates forboth. The target translocation rate was then normalized with thetranslocation rate of the control molecule.

Table 1 shows the normalized sample rates of these target moleculesafter further normalization to the dA₉dA₂₄ (SEQ ID NO: 12) rate. Theseare the concentration enhancement factors that indicate the relativeincrease in sample translocation rates by incorporation of thehydrophobic capture element compared to those without. It is noted thatthe concentration enhancement factors are less in Table 1 than thoseshown in FIG. 4A. In this regard, it is believed that the Table 1 datawere better controlled for concentration which likely accounts for thediscrepancy. However, the data presented in both FIG. 4A and Table 1illustrate significant enhancement in translocation rates.

TABLE 1 dA₉dA₂₄ dA₆C₃₆dA₂₄ dA₅C₄₈dA₂₄ dA₄C₆₀dA₂₄ C₁₀₈dA₂₄ SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID NO: 12) NO: 8) NO: 9) NO: 10) NO: 11) SampleRate 1 30.9 28.8 26.5 12.5 (Normalized)

The leader length that extends beyond the hydrophobic capture elementmay also be modified for interaction with the nanopore. To this end, theleader should be of a sufficient length such that its capture in thenanopore exerts enough force to uncouple the target molecule from thebilayer or, depending on the embodiment, unlink the leader/targetportion from the hydrophobic capture element. The leader should carryelectrostatic charge to promote interaction with the nanopore under anapplied electric potential. A nucleic acid is typically anionic and theleader would typically also be anionic. In some cases an end portion ofthe target portion may also function as the leader. The leader istypically a single linear polymer, but may have two or more linearpolymer portions to help improve nanopore interaction, and should alsobe able to translocate the nanopore so the target molecule can thenengage. Leader materials can be synthesized from many anionic, cationicor neutral polymers and may be made of combinations of materials such as(but not limited to) heterogenous or homogeneous polynucleotides,polyethylene glycol, polyvyinyl alcohol, polyphosphates,poly(vinylphosphonate), poly(styrenesulfonate), poly(vinylsulfonate),polyacrylate, abasic deoxyribonucleic acid, abasic ribonucleic acid,polyaspartate, polyglutamate, polyphosphates, and the like. For example,a representative leader may comprise PEG-24 and/or poly-A₁₂.

The effect of leader length upon translocation rates was compared bymodifying a target with different length leaders that extend beyond thehydrophobic capture element (C48). The same control and target moleculesshown in FIG. 5 were used with the end modifications (Y^(3′)) shown inFIG. 6. Normalized translocation rate results are shown in Table 2 andare identified as dA₁₈C₄₈dA₁₁ (SEQ ID NO: 16), dA₁₁C₄₈dA₁₈, (SEQ ID NO:15), dA₅C₄₈dA₂₄ (SEQ ID NO: 14) and dA₅C₄₈dA₂₄L₂₅ (SEQ ID NO: 13). Allnanopore measurements supporting the results in Tables 1 and 2 used thesame experimental conditions and used the same control molecule so theycan be directly compared. For this reason, results in both tables arenormalized to the dA₉dA₂₄ (SEQ ID NO: 12) result of Table 1, thusreferencing the enhancement in translocation rate (also referred to asconcentration enhancement herein) to a similar molecule with nohydrophobic capture element.

TABLE 2 dA₉dA₂₄ dA₁₈C₄₈dA₁₁ dA₁₁C₄₈dA₁₈ dA₅C₄₈dA₂₄ dA₅C₄₈dA₂₄L₂₅ (SEQ ID(SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 12) NO: 16) NO: 15) NO: 14) NO: 13)Sample Rate 1.0 2.8 10.7 28.8 49.0 (Normalized)

These results indicate that the concentration enhancement factorincreases as the polyA leader increases from 11 to 24 bases. In anothermeasurement, using a different target molecule, the influence of endgroups dA₅C₄₈dA₂₄ (SEQ ID NO: 9) and dA₅C₄₈dA₅₀ (SEQ ID NO: 28) werecompared. This showed the latter (longer) leader to be 82% of the formerindicating the enhancement effect of polyA leaders plateaus in the rangeof 20 to 50 bases.

The last column of Table 2, shows the enhancement result due to a dA₂₄leader that is extended with a 25 ethyl phosphodiesters (dA₅C₄₈dA₂₄L₂₅)(SEQ ID NO: 13). Its concentration enhancement factor was 70% largerthan dA₅C₄₈dA₂₄ (SEQ ID NO: 14) alone. Additional leader measurementsare presented in Table 4.

The hydrophobic capture element is designed to promote mobility in thelipid bilayer and maintain the hydrophobically captured state, butlimited enough so that the target can be released when interacting withthe nanopore. The element can extend the target backbone and be in-linewith the leader or may be pendant to the backbone or may have multipleelements pendant to the backbone. The hydrophobic capture element can bepositioned anywhere along the target relative to the leader but can beoptimized to improve capture by the nanopore. Materials that comprisethe hydrophobic capture element include, but are not limited to, linearand branched aliphatic chains, lipids, fatty acids, DBCO, cholesterol,fluorinated polymers, apolar polymers, steroids, polyaromatichydrocarbons, hydrophobic peptides, and hydrophobic proteins. This mayalso include phase transition polymers that can switch from hydrophilicto hydrophobic states under thermal or other environmental change. Insome embodiments some or all of the heads of the lipids in a bilayer arereactive and can bind to an adapted target molecule as shown by Grenaliet al. (supra). In this case, the lipid is the hydrophobic captureelement.

The method variation shown in FIG. 3 was demonstrated using the targetsend-adapted with either dA₉dA₂₄ (SEQ ID NO: 12) or dA₅C₄₈dA₂₄ (SEQ IDNO: 14) and the control shown in FIG. 5. In this example, a hydrophobiccapture element was used that has a C120 on one end (synthesized bylinking ten, C12 monomers), and an oligomer at the other end. Thisoligomer was complementary to a nucleic acid region at the end of thetarget adjacent to the end modification. FIG. 7 shows several differentversions of this hydrophobic capture element. For each measurement, a 2microliter aliquot of 300 femtomoles of a capture element was injectedinto the cis reservoir adjacent to the nanopore. The hydrophobic C120group on the capture element is inserted into the lipid bilayer, withits other oligomer end remaining outside the bilayer in the aqueousbuffer. The cis reservoir was then exchanged with fresh buffer and a 2microliter aliquot of sample was added containing 15 femtomoles oftarget and 15 femtomoles of control. The target molecule can hybridizeto the capture element and diffuse along the plane of the bilayer. Incontrast the control shown in FIG. 5 has no complementary region andwill not hybridize to the capture elements described in FIG. 7. Nanoporetranslocation begins when the leader is electrophoretically pulledthrough and stops at the capture element duplex. The duplex releases dueto thermal and electrophoretic pulling forces, allowing translocation toproceed.

Table 3 shows concentration enhancement factors for these molecules(normalized to the target with no hydrophobic capture element; dA₉dA₂₄(SEQ ID NO: 12). All measurements were made under the same conditionsand concentrations described above. Note that molecules adapted withdA₅C₄₈dA₂₄ all have a second hydrophobic capture element. Comparing thetwo CE1 results indicates that having this second hydrophobic captureelement increases the concentration enhancement factor. Reducing theduplex length from 16 bases (CE1) (SEQ ID NO: 17) to 11 bases (CE2 (SEQID NO: 18), CE3 (SEQ ID NO: 19) and CE4 (SEQ ID NO: 20)), reduces thestability and enhancement is decreased. The CE2 (SEQ ID NO: 18) and CE3(SEQ ID NO: 19) capture elements had similar structure except the Cl 20hydrophobic group was positioned on opposite ends of the duplex. CE4(SEQ ID NO: 20) had 5 PEG-6 spacers between the hydrophobic group andthe hybridization site and improved the concentration enhancementrelative to both CE2 (SEQ ID NO: 18) and CE3 (SEQ ID NO: 19), which isbelieve to be due to relaxing how tightly the duplex was held to thelipid bilayer.

TABLE 3 dA₉dA₂₄ dA₅C₄₈dA₂₄ (SEQ ID NO: 12) (SEQ ID NO: 14) CaptureElement None CE1 CE1 CE2 CE3 CE4 Sample Rate 1 417 890 15 100 298(Normalized)

Additional target molecules were tested that were short ds-DNA strandsshown in FIG. 8. Unlike the measurements for results in Tables 1, 2 and3, these targets had only a single duplex and had no control added tothem for normalization. Otherwise the measurement conditions were thesame. Each was measured with a 30 femtomole sample and all werenormalized to C₄₈A₂₄ (SEQ ID NO: 21). This indicates that dL₂₄ enhancescapture by the nanopore more than dA₂₄ by a factor of 2.8, and thatadding longer extensions of L₁₀₀ provides even greater enhancement.

TABLE 4 C₄₈dA₂₄ C₄₈L₂₄ C₄₈dA₂₄L₁₀₀ (SEQ ID (SEQ ID (SEQ ID NO: 21) NO:22) NO: 23) Sample Rate 1.0 2.8 3.7 (Normalized)

In addition, the surface can be modified to optimize performance of thehydrophobic capture element. For example, when the surface is a lipidbilayer, increasing mobility of the captured target molecule increasesthe probability of leader interaction with the nanopore. For example,increasing the area of the lipid bilayer increases the probability thatthe target molecules will be captured and migrate to the nanopore.Target molecule capture in the bilayer may also be improved byminimizing any undesired trapping on undesired surfaces in thereservoir, such as isolated lipid or non-lipid reservoir walls. The useof tethered bilayers is a powerful design tool that could be used tocontrol the relative mobility and capture kinetics of the bilayersurfaces. Utilizing the characteristics of fixed lipids and lipidadditives to define these characteristics, the target molecules can becaptured and limited to diffuse in preferred directions along thebilayer surface. For example, by constraining the lipid layer to be along thin rectangle confines any hydrophobically captured molecules todiffuse principally along its length.

FIG. 9 depicts a supported lipid bilayer (901) used in conjunction witha solid-state nanopore (904). FIG. 9 is similar to the embodimentdepicted in FIG. 3, and depicted in this manner (as opposed to theembodiment of FIG. 1 or 2) for purpose of illustration only. Referringto FIG. 9, supported lipid layers are synthesized using a tether species(906) that covalently bond to substrate (908) at one end and imbeds intoa bilayer on the other end (see J. Jackman et al., “BiotechnologyApplications of Tethered Lipid Bilayer Membranes,” Materials5(12):2637-2657, 2012). A common inorganic film used for solid-statenanopores is silicon nitride which can oxidize to form silicon oxide onits surface. Atanasov et al. has shown supported lipid bilayer formationtether-stabilized with lipids adapted with silanes to bond to a siliconoxide surface (“Membrane on a Chip: A Functional Tethered Lipid BilayerMembrane on Silicon Oxide Surfaces,” Biophys J., 89(3):1780-1788, 2005).These bilayers maintain the required diffusion characteristics thatenable the hydrophobically captured molecule to migrate near thenanopore. This bilayer does not need to maintain high electricalimpedance, but does require that the bilayer integrity be sufficientnear the nanopore such that the target molecule leader can be captured.

Additional forces can be applied to the hydrophobically associatedtarget molecules that will steer them in a preferred direction along thelipid bilayer or other hydrophobic/hydrophilic interface. Graneli et al.(“Organized Arrays of Individual DNA Molecules Tethered to SupportedLipid Bilayers,” Langmuir 22(1):292-299, 2006) demonstrated that DNAlinked to the head group of a lipid that was in a supported lipidbilayer could be moved laterally by the flow of the buffer across thebilayer. Furthermore the DNA-tethered lipid would stop at a defineddiffusion barrier, fixing that end of the DNA while the flow remained.After flow was stopped, this lipid molecule and its tethered DNA woulddiffuse away from the barrier along the bilayer membrane.

FIGS. 10A and 10B show a supported lipid bilayer (1001) in a shapedefined by the diffusion barrier at its edges (1003). Arrow (1005) showsthe direction that buffer above the bilayer is flowing. This flowingbuffer applies a shear force to the target molecules (1007) that dragsthem along until they are interrupted by the diffusion barrier (FIG. 10Arepresents the location of the target molecules before application ofthe shear force, while FIG. 10B represents location of the targetmolecules after application of the shear force.) By angling thesebarriers relative to the flow direction, the target molecules (1007) areconcentrated in an area in proximity to nanopore (1008) (see FIG. 10B).This technique can be used to collect and concentrate target moleculesin low concentration from larger volumes near a nanopore (or eachnanopore in a nanopore array). In addition to flow, other forces can beemployed to move the target molecules along the bilayer surface,including electrophoretic/electroosmotic forces (C. Liu et al. “ProteinSeparation by Electrophoretic-Electroosmotic Focusing on Supported LipidBilayers,” Anal. Chem. 83(20):7876-7880, 2011.), and acoustic forces (J.Neumann et al., “Transport, Separation, and Accumulation of Proteins onSupported Lipid Bilayers,” Nano Lett. 10(8):2903-2908, 2010).

The method of this invention may be modeled with reservoir targetmolecule concentration N_(R) and rate constants for:

-   -   i) capture of the leader by the nanopore from the bilayer        (k_(B-trans)),    -   ii) capture of the leader by the nanopore from the reservoir        (k_(R-trans)),    -   iii) capture of hydrophobic group in the bilayer (k_(Bcapt)),    -   iv) passive release of hydrophobic group from the bilayer        (k_(Brel)),

In this model, the reservoir may be considered infinite and N_(R)constant. The rate of translocations of molecules pulled directly fromthe reservoir is:{dot over (N)} _(R-trans) =k _(R-trans) N _(R),

Along the hydrophobic capture path, the surface concentration ofmolecules (associated with in the bilayer), N_(B), changes as:{dot over (N)} _(B) =k _(Bcapt) N _(R)−(k _(Brel) +k _(B-trans) /A)N_(B) when k _(B-trans) N _(B) /A<N _(Bsaturation)Note that this simplified equation has factor of lipid area, A, that isinserted to normalize the rates of molecule capture/release across alipid area with the molecules translocating thru a single nanopore onthe area. This assumes that molecular depletion from the lipid (due totranslocation) happens uniformly across A.

At steady-state:0=k _(Bcapt) N _(R)−(k _(Brel) +k _(B-trans) /A)N _(B)N _(B) =k _(Bcapt) N _(R)/(k _(Brel) +k _(B-trans) /A)

Choosing area, A, sufficiently large where A>>k_(B-trans)/k_(Brel),leads to:N _(B) =k _(Bcapt) N _(R) /k _(Brel)

A strong hydrophobic group leads to k_(Bcapt)/k_(Brel)>>1 which leads tohigh surface concentration of target molecules tethered to the lipiddespite relatively low concentration of molecules in the reservoir.

The translocation rates k_(B-trans) and k_(R-trans) are related butdiffer by the following factors:

-   -   i) mobility of target molecule on the lipid surface vs mobility        in the reservoir.    -   ii) effective translocation capture cross-section of molecule        end as a function of distance from nanopore. Note that surface        tethered case has additional factors to this including position        of hydrophobic group and length of the leader.

The rate of translocation can have several regimes including:

-   -   i) Diffusion-limited: In this case the molecules must diffuse so        their capture end is within range of the nanopore.    -   ii) End-capture limited: In this case, many molecules are within        range (up to the maximum concentration) and translocation rate        is limited by the time it takes to capture the end of one of        these molecules.    -   iii) Translocation-limited: In cases where only 1 or some        limited number of molecules can enter the nanopore, other        molecules can be within range but must wait until the nanopore        is available for translocation of another molecule.

Example 1 Nanopore Measurement of Target with C48 Capture Element andPolyA₂₄ Oligomer Leader

Target molecule synthesis is performed using a Mermaide 4oligonucleotide synthesizer (BioAutomation, Texas) using commerciallyavailable amidites (Glen Research, Sterling, Va.; Chem Genes,Wilmington, Mass.). The following target molecules are synthesized:

Target 1 - (SEQ ID NO: 29) (dA)₂₄(dCdGdGdGdCdAdAdTdAdA dGdCdCdC);Target 2 - (SEQ ID NO: 30) (dA)₂₄ (Dodecyl phosphodiester)₄(SEQ ID NO: 31) (dA)₅ (dCdGdGdGdCdAdA dTdAdAdGdCdCdC);Each target molecule was page purified on a 6% acrylamide TBE-Urea gel(Life Technologies, Carlsbad, Calif.). Both target molecules contain apoly dA leader portion and a stem-loop structure, which is used tocontrol translocation speed and direction. Target 2 includes theaddition of four dodecyl phosphodiester linked monomers, which createthe C48 capture element. Each purified target molecule is analyzed usingthe α-hemolysin nanopore system described by Jetha et al. (Chapter 9.Micro and Nano Technologies in Bioanalysis, Humana Press 2009). Targetsare added to the cis reservoir of the nanopore device that contains 100ul 2.0 M LiCl, 10 mM HEPES, pH of 7.4. The trans reservoir contains thesame solution. Event frequencies are determined for each target across arange of target inputs (1 fmole to 1 pmole) and voltages (100-140mVolts) to determine the concentration effect of the C48 captureelement.

Example 2 Nanopore Array Capturing Rare Nucleic Acid Targets withConcentrator

Detection and identification of nucleic acids at very low concentrationis generally not practical without molecular amplification. Bypresenting a thin film of sample across a large nanopore sensor array,the target molecules can diffuse to the sensor surface in reasonabletime periods. If the sensor surface is primarily a hydrophobic domain,target molecules modified with at least one hydrophobic capture elementassociate and diffuse along the surface. This greatly increases thelikelihood of being sensed.

A microfluid flow cell is designed with a chamber through whichelectrolyte with sample can pass through. The chamber is 100 microns inheight, 3 mm wide and 10 mm long with 1.0 mm diameter input and outputports located at the ends on the top side. On the top side is glass orpolymer that is surface treated to inhibit binding to nucleic acids. Thebottom side is sealed against a silicon chip that contains a 200×500array of nanopore cells. The array lies on a grid with 15 microncenters. The outer dimension of the array is 3 mm×7.5 mm and is centeredin the chamber. Each cell contains a shallow 10 micron diameter by 3micron deep well that has an Ag/AgCl electrode at its base. Theelectrode passes current from contacting electrolyte to be measured bythe nanopore cell's transconductance amplifier. The current-convertedvoltage outputs from the array of nanopore amplifiers are measured atbandwidths exceeding 1 ksample/s/cell.

Across the surface of the silicon chip exposed to the flow chamber, acontinuous lipid bilayer is formed in an electrolyte buffer. It issuspended as a membrane over each cell well but is a supported lipidbilayer over the remaining area. Hemolysin nanopores are inserted intothe bilayer in a manner to maximize the number of wells with singlenanopores. The lipid layer that is connected to the substrate is formedso as to electrically isolate adjacent cell wells from current passingbetween the substrate and the bilayer. This isolation is sufficient thatany leakage currents can be ignored compared to currents that passthrough the single nanopore. A characteristic of the continuous bilayeris that molecules adapted with a hydrophobic group as described hereincan associate with the bilayer from the flow chamber and will diffuseanywhere along its surface.

A pathogen assay uses hybridization and ligation specificity to identifyDNA by using the DNA as a template to hybridize and ligate the targetshown in FIG. 5 with dA₅C₄₈dA₂₄L₂₅ (SEQ ID NO: 13) as shown in FIG. 6using methods as taught depicted in U.S. Pat. No. 8,586,301. A 3microliter sample of this ligation product is injected microfluidicallyto fill the chamber where the ligation products can diffuse until theycontact and associate with the lipid bilayer (due to the C₄₈ group). Theligation products then diffuse along the plane of the lipid bilayeruntil they are captured and measured in a nanopore. This method ishighly sensitive because for a 3 microliter sample, all volume diffusingtargets are localized to within 100 microns of the active surface,surface-diffusing targets are localized to within 10 microns of ananopore and measurements provide target specific information from asingle molecule.

In alternative embodiments, the geometry described above can be modifiedin a variety of ways, including (for example) the modifications notedbelow.

(i) To inspect larger volumes of sample the chamber and lipid bilayercapture surface can be extended upstream. With suitable diffusionbarriers in the lipid, flow induced concentration as described usingFIGS. 10A and 10B can be used to collect the targets downstream at thenanopore array.

(ii) Provided the target concentration is uniform in chamber volume, itwill collect uniformly at each well. In this case the lipid bilayer needonly be continuous over each well. The electrical isolation of each wellcould coincide with a break in the lipid layer. To maintain highcollection efficiency, the area of the bilayers (that collect and alongwhich target molecules can diffuse) should be as large as possible.

(iii) By adapting the top surface of the flow chamber to have anotheractive silicon chip reduces the average diffusion distance that theinjected target must diffuse to reach a bilipid layer and reducessurface area that can lead to sample loss.

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent applications, foreign patents, foreign patentapplications and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet, including butnot limited to U.S. Pat. No. 7,939,259, are incorporated herein byreference, in their entirety. Aspects of the embodiments can bemodified, if necessary to employ concepts of the various patents,applications and publications to provide yet further embodiments.

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

The invention claimed is:
 1. A method for concentrating target molecules for nanopore sensing, comprising: capturing target molecules on a surface in contact with a cis reservoir, wherein the surface comprises a nanopore penetrating through the surface and in fluid communication with a trans reservoir on an opposing side of the surface, and a hydrophobic domain, the hydrophobic domain comprising a diffusion barrier surrounding the nanopore and defining a shape selected for concentrating the captured target molecules in proximity to the nanopore upon flow of a buffer across the surface, wherein each of the target molecules comprises a target portion, a hydrophobic capture element and a leader for interaction with the nanopore, and wherein the hydrophobic capture elements are associated with, and capable of movement along, the hydrophobic domain of the surface to bring the leaders in proximity with the nanopore upon flow of a buffer across the surface; flowing a buffer across the surface to concentrate the target molecules in proximity to the nanopore, applying an electric field across the nanopore, thereby causing the leaders of the captured target molecules to interact with the nanopore, and the target portion to dissociate from the surface and translocate through the nanopore; and sensing at least the target portion upon translocation through the nanopore.
 2. The method of claim 1, wherein the step of capturing the target molecule on the surface comprises contacting the surface with the target molecule, wherein the target molecule comprises, prior to the contacting step, the target portion, the hydrophobic capture element and the leader.
 3. The method of claim 1, wherein the step of capturing the target molecule on the surface comprises linking the hydrophobic capture element associated with the surface to the target portion and leader, thereby capturing the target molecule on the surface.
 4. The method of claim 1, wherein the nanopore is a biological nanopore.
 5. The method of claim 1, wherein the surface is a lipid bilayer.
 6. The method of claim 1, wherein the surface is a solid-state or synthetic membrane.
 7. The method of claim 1, wherein the target portion comprises nucleic acids.
 8. The method of claim 1, wherein the target portion comprises a linear polymer.
 9. The method of claim 1, wherein the target portion comprises a molecular bar code.
 10. The method of claim 1, wherein the target portion comprises linkers and a nucleotide sequence complementary to a target nucleic acid sequence, wherein the combined linear length of the linkers and nucleotide sequence is longer than the linear length of the target nucleic acid sequence.
 11. The method of claim 1, wherein the leader is a hydrophilic polymer.
 12. The method of claim 1, wherein the hydrophobic capture element is an aliphatic hydrocarbon.
 13. The method of claim 1, wherein the target molecules comprises two or more hydrophobic capture elements. 